HIV, the causative agent of AIDS, is a complex retrovirus. Tat and rev are required for virus replication in all settings. The vif, vpr, vpu, and nef genes are generally not required for viral replication in vitro. In vivo, however, they all act together as important determinants of virulence, although their individual contributions are not always obvious. Even in animal models that simulate the complex pattern of pathology associated with AIDS, the roles of some of these genes are unclear. Nef, for example, which has been shown to virtually have an all-or-nothing effect in the development of an AIDS-like syndrome in adult macaques, is hot required to induce disease in neonates. Considerable evidence indicates that the levels of HIV/SIV replication largely determine the final outcome of the disease and that inhibition of virus replication results in significant benefits to infected patients. We hypothesize that inhibition of Nef function can significantly reduce virus replication in vivo and that such inhibition will positively affect the final outcome of infection. Even if these benefits do not extend to neonates, the majority of HIV-1 positive adults are likely to benefit from Nef inhibitors. In addition, drugs targeting Nef could act prophylactically to prevent the establishment of HIV-1 infection. Thus, the focus of this competitive renewal grant is to implement a novel in vivo system of HIV infection developed in our laboratory to begin to dissect the individual contributions of the different in" vitro activities of HIV-1 Nef to pathogenesis and disease progression. This in vivo system consists of a humanized mouse model system that replicates key features of the human immune system and HIV-1 infection. Effective utilization of this model is dependent on mechanistically interpretable mutations of nef incorporated into HIV-1. Advances in this regard have been achieved by precise analysis of Nef structure/function based on the well documented conservation of subtype B Nef. Based on these advances we propose to evaluate the overall importance of Nef in HIV pathogenesis and disease progression, and the role of individual Nef functions. Our specific aims are: 1) To determine the role of Nef in the pathogenesis associated with HIV-1 infection of humanized BLT mice. 2) To determine the contribution of CD4 downregulation by HIV-1 Nef to pathogenesis. 3) To determine the role of Pak2 activation by Nef in HIV-1 pathogenesis in vivo.